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Mycobacterium tuberculosis specific fusion protein vaccine ab and its preparation and application

A technology of Mycobacterium tuberculosis and fusion protein, which is applied in the new type of Mycobacterium tuberculosis-specific fusion protein vaccine AB and application fields, can solve the problems of mouse death and hypersensitivity reactions, and achieve enhanced cellular immune function and high expression level. , the effect of increasing production

Active Publication Date: 2015-09-16
THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above two recombinant protein vaccines have entered phase I and II clinical trials, but foreign patented products, and wherein ESAT6 is a protective factor of Mycobacterium tuberculosis, but also a virulence factor, the inventor's research shows that ESAT6DNA immune tuberculosis Infected individuals, potentially causing hypersensitivity reactions leading to death in mice

Method used

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  • Mycobacterium tuberculosis specific fusion protein vaccine ab and its preparation and application
  • Mycobacterium tuberculosis specific fusion protein vaccine ab and its preparation and application
  • Mycobacterium tuberculosis specific fusion protein vaccine ab and its preparation and application

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preparation example Construction

[0053] The above-mentioned preparation method of fusion protein AB comprises:

[0054] (1) Design of the fusion of two protein antigen epitopes: analyze the gene sequence and protein structure of Mycobacterium tuberculosis Ag85A and Ag85B, determine the fusion region, combination and sequence of the two protein antigen epitopes; design A and B protein antigens Epitopes are connected in sequence to form a fusion protein; the antigenic epitope of protein A is located at the amino terminal of the fusion protein, and the antigenic epitope of protein B is located at the carboxyl terminal of the fusion protein.

[0055] (2) Cloning of fusion of two proteins: cloning by genetic engineering technology.

[0056] ① Add an Nde I restriction site to the upstream primer of the protein A coding gene, add an Xho I restriction site to the downstream primer of the protein A coding gene, double digest the PCR amplification product with Nde I and Xho I, and insert In the pET-30a plasmid vector ...

Embodiment 1

[0060] 1. Cloning the gene encoding protein A epitope by genetic engineering technology:

[0061] 1. Design and synthesize a pair of primers for amplifying the A antigen epitope according to the sequence 1 in the sequence listing

[0062] Upstream primer (5' end contains restriction endonuclease Nde I, underlined)

[0063] 5'-GCAATTC CATATG TTTTCTCGT-3'

[0064] Downstream primer (5' end contains restriction endonuclease Xho I, underlined)

[0065] 5'-CCG CTCGAG TTTGAGAAAGG-3'

[0066] Amplified fragment: 931bp

[0067] 2. Synthesis of A protein coding gene

[0068] According to the above design, the PCR product of A protein coding gene 931bp was synthesized by gene synthesis, and the synthesized gene fragment was subjected to sequence analysis (T7 and T7-ter universal primer bidirectional sequencing verification), and the sequencing results proved that the PCR product of gene synthesis was A Protein-coding genes, the specific results are as follows.

[0069] The desi...

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Abstract

The invention relates to a mycobacterium tuberculosis specific fusion protein vaccine AB and preparation and application thereof. The fusion protein vaccine AB is formed by sequentially connecting Ag85A protein epitope and Ag85B protein epitope; and the amino acid sequence of the protein vaccine AB is shown by the sequence 3 in a sequence table. The AB / pET-30a recombinant plasmid is established from the Ag85A protein epitope and Ag85B protein epitope by the genetic engineering technology and then transferred into escherichia coli, and the expressed fusion protein is formed by sequentially connecting the epitopes of the proteins A and B. The mycobacterium tuberculosis specific fusion protein vaccine AB provided by the invention can obviously enhance the mouse specific cellular immune function and is mainly used for irritating the Th1 type immune reaction; and a mouse tuberculosis treating model can remarkably reduce the bacterial population of the organs, and can be used for preparing a medicine or vaccine for preventing or treating tuberculosis.

Description

technical field [0001] The invention relates to a Mycobacterium tuberculosis-specific fusion protein vaccine AB and its preparation and application, in particular to a novel Mycobacterium tuberculosis-specific fusion protein vaccine AB prepared by applying genetic engineering technology and its application, belonging to tuberculosis medical immunology Therapeutic technology field. Background technique [0002] Tuberculosis (Tuberculosis, TB) has been a global health issue of concern. About 1 / 3 of the world's population is infected with Mycobacterium tuerculosis (Mtb), with 8-10 million new cases of tuberculosis and more than 2 million deaths from tuberculosis each year (World Health Organization 2006 Tuberculosis Facts). The number of tuberculosis patients in my country ranks second in the world, second only to India. The fifth national tuberculosis epidemiological survey in 2010 showed that there are currently 5-6 million tuberculosis patients in my country, more than 1.4...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70A61K39/04A61P31/06
Inventor 吴雪琼梁艳张俊仙阳幼荣
Owner THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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